1. Field of the Invention
This invention relates to a mutant which is resistant to a cell membrane synthesis inhibitor, and, more particularly, to a cell membrane synthesis inhibitor-resistant mutant of an extracellular enzyme-producing microorganism belonging to genus Bacillus, such as cellulase-, protease-, or amylase-producing microorganism belonging to genus Bacillus. The invention also relates to a process for preparing such a mutant.
2. Description of the Background
Cellulase is realized as a group of complicated enzymes catalyzing enzymatic reactions decomposing cellulose into glucose, cellobiose, or even into a cello-oligosaccharide. Depending on the functions, it encompasses enzymes such as C.sub.x cellulase (CMC-ase or endo-.beta.-1,4-glucanase), C.sub.1 cellulase (cellobiohydrolase or exo-.beta.-1,4-glucanase), and .beta.-glucosidase (cellobiose .beta.-1,4-glucosidase), and the like.
Conventionally, studies on cellulase have been directed, for the most part, to the effective utilization of biomass resources. For this reason major supply sources of cellulase have been fungus belonging to genera such as Trichoderma, Aspergillus, Acremonium, Humichola, and the like.
Recent development of a new application of cellulase as an additive to detergents for washing clothes is requiring supply of alkaline cellulase having a better stability and exhibiting maximum activity at a pH of alkaline side at which cloth washing is performed with a better efficiency.
There have been known several processes for producing alkaline cellulase. Until recently, such processes have been typified by a process for collecting cellulase A from a culture broth of an alkalophilic microorganism belonging to genus Bacillus (Japanese Patent Laid-open No. 28515/1985), a process for producing alkaline cellulase 301-A through culturing of a microorganism belonging to genus of Cellulomonas (Japanese Patent Laid-open No. 224686/1983), a process for obtaining carboxymethyl cellulase through culturing of an alkalophilic strain, Bacillus sp. no. 1139 [Fukumori F., Kudo T., and Horikoshi K.; J. Gen. Microbiol., 131, 3339 (1985)], a process for producing alkaline cellulase with the use of a microorganism belonging to Streptomyces (Japanese Patent Laid-open No. 19483/1986). Micrcorganism used in these processes had only a low alkaline cellulase productivity and thus the processes have not been suitable for industrial application.
In view of this situation the present inventors have undertaken extensive studies in order to obtain a micrcorganism having a better alkaline cellulase productivity. As a result, inventors have previously discovered several novel microorganisms having superior alkaline cellulase producing capability, including Bacillus sp. KSM-635 (FERM 8872), and filed patent applications on these microorganisms (Japanese Patent Application No. 257775/1986 and others).
Formulating protease into detergents have long been practiced. Earlier practice was to formulate into detergents protease having an optimum pH close to the neutral region such as papain, pancreas trypsin, or the like. Like cellulase, however, protease having an optimum pH in an alkaline side and stable in surface active agents have been desired for the detergent use. Development of such protease has been undertaken.
Typical alkaline proteases used for detergent purpose are alkalase, savinase, esperase (manufactured by Novo Industries Co.), maxatase (produced by Gist Brocades Co.), and the like. The optimum reaction temperature for these enzymes is in the neighborhood of 60.degree. C. and is in conformity with the washing practice of some region, for example, European countries and U.S.A.
However, in other regions where washing is performed near room temperature, supply of protease are desired which has a strong activity at a temperature lower than that at which conventional protease are active. Furthermore, because of common knowledge that a protease with a superior capability of decomposing insoluble proteins such as keratin can contribute to detergency [Minagawa M.; Sensho-shi (J. Jpn. Res. Assn. Text. End-Uses, 26, 322 (1985)], the desire has existed for a protease having a superior insoluble protein decomposing capability.
The present inventors have carried out extensive studies in order to obtain an alkaline protease having keratin-decomposing capability, and a lower optimum temperature, i.e., exhibiting high activity at a low temperature, and discovered a group of microorganisms, including Bacillus sp. KSM-2001 (FERM P-9449), satisfying these requirements. The inventors previously filed a patent application (Japanese Patent Application No. 261487/1987) based on these findings.
Attention has been given also to amylase capable of hydrolyzing starch as a component of detergents, since starch are abunduntly contained in the dirt of foods and the like. Examples of known amylases are neutral amylase produced, for example, by Bacillus amyloliquefaciens [N. E. Welker and L. L. Campbell; J. Bacteriol., 94, 1124, (1967)], heat resistant amylase produced, for example, by Bacillus licheniformis (Japanese Patent Laid-open No. 37094/1986), alkaline amylase produced, for example, by Bacillus ohbensis (Japanese Patent Publication No. 31949/1977), and the like.
Development of the above-mentioned new microorganisms has made it possible to produce extracellular enzymes for practical application. For the purpose of industrial production, however, a need has continued to exist for the further development of extracellular enzyme-producing microorganisms other than those mentioned above, as well as improvement in productivity of extracellular enzyme by microorganisms.
In this situation the inventors have undertaken continued studies for the improvement of productivity of extracellular enzymes, especially by means of mutation, and found that a remarkable productivity improvement was attained by providing extracellular enzyme-producing microorganisms belonging to genus Bacillus with resistance to antibiotics cell membrane synthesis inhibitors. This finding has led to the completion of this invention.